Gene amplification technology and PCR method
A technology to amplify nucleic acids such as DNA and RNA has been widely disseminated in both basic research and industrial application along with development of gene engineering technology. At an early stage, in order to obtain a particular target nucleic acid sequence, the sequence were in general prepared by cutting out the necessary sequence with restriction enzymes from nucleic acids amplified in yeast or Escherichia coli cells. Subsequently, it became possible to amplify a target particular nucleic acid region in vitro by the development of PCR by Mullis et al and dissemination thereof. For the PCR method, various experimental and industrial applications have been developed in parallel with active commercialization and distribution of specific apparatuses and related reagents. Thus the PCR method became the substantial standard method for amplifying the gene. As the research for the DNA replication has advanced, various technologies whose principals are different from that of PCR have been designed and developed, but they are less common in terms of operationality, cost and quality, and no technology which sweeps aside the PCR method has appeared.
Points for Evaluating PCR Method
The principle of the PCR method is thought to be based on minimally mimicking the intracellular DNA replication. That is, the principle is based on (1) dissociation of a target nucleic acid template by thermal denaturation, (2) pairing of the target sequence with a pair of complementary primers and (3) extension of the primer complementary to the template by DNA polymerase. The objective nucleic acid sequence is exponentially amplified by repeating these reactions continuously. In a general protocol of PCR, several kb of the target sequence in the template in nano gram order is often amplified to obtain a product in μg order by the reaction for about two hours.
The PCR method has technical restrictions, which representatively include the following 4 points: (1) fidelity to the template (performance to amplify the sequence precisely corresponding to the template), (2) extendibility (performance to amplify the longer sequence), (3) efficiency to amplify the target sequence, and (4) reaction specificity. These are also often addressed as the points which evaluate PCR. Reagents and kits for PCR have been being developed for the purpose of overcoming these restrictions.
Improvement of PCR
As DNA polymerase for PCR, DNA polymerase derived from a thermophilic bacterium Thermus.aquaticus (Taq DNA polymerase) used to be generally used in the initial period (Non-patent Document 1). Based on this enzyme, subsequently various enzymes and reagents/kits for PCR have been developed and distributed. Most of them have an improved feature on any of the aforementioned technical restrictions, and the reagents/kits having such characteristics have been distributed by various manufacturers. Some examples of the improved PCR methods are shown below.
As an example of enhancing the fidelity to the template, DNA polymerase with high fidelity is generally used for the PCR method. Taq DNA polymerase is a PolI type DNA polymerase having only 5→-->3′ polymerase activity and having no 3′-->5′ exonuclease activity. On the contrary, DNA polymerase derived from an ultrathermophilic archaebacterium, Pyrococcus.furiosus is an α type DNA polymerase which has both the 5′-->3′ polymerase activity and the 3′-->5′ exonuclease activity. The 3′-->5′ exonuclease activity works as a proof reading activity. Thus, when this DNA polymerase is used for PCR, the fidelity to the template upon amplification is strikingly enhanced compared to amplification with Taq DNA polymerase having no 3′-->5′ exonuclease activity (Non-Patent Document 2).
Examples of commercially available products among such DNA polymerases may include Pyrobest DNA polymerase (TAKARA BIO INC.), Pfu DNA polymerase (Stratagene), KOD DNA polymerase (Toyobo Co., Ltd.), DeepVent DNA polymerase (New England Biolabs (NEB)), Vent DNA polymerase (NEB) and Pwo DNA polymerase (Roche Diagnostics).
Barns et al. has reported that when the a type DNA polymerase having the proof reading function and the PolI type DNA polymerase having no proof reading function were mixed at an appropriate ratio and used for PCR, the length of the extendable target sequence was increased and the amplification efficiency was also enhanced (Non-patent Document 3, Patent Document 1). Examples of the commercially available products of such mixed DNA polymerases may include TaKaRa EX Taq DNA polymerase (TAKARA BIO INC.) and Taq Plus Long (Stratagene).
As one example of the strategy directing to enhancement of the reaction specificity of PCR, a hot start method has been known. Non-specificity in PCR is often caused by non-specific annealing of the primer to the template DNA. To prevent this phenomenon, the hot start method is effective in which the PCR is started immediately after completely mixing a PCR reaction solution at high temperature. Several methods for the hot start method have been reported. In a current mainstream, DNA polymerase is complexed with its specific antibody and inactivated at low temperature, and the DNA polymerase is activated under a high temperature condition upon hot start to initiate the PCR reaction. It has been reported that the specificity of the PCR reaction is enhanced by this method (Patent Document 2). Products associated with this hot start method are also distributed by gene engineering manufacturers, and generally utilized.
The aforementioned methods for improving the PCR method all have come into practical use, commercialized and generally used, but all have both advantages and disadvantages, and do not satisfy all the points for improvement in the PCR method discussed in the above. For example, the α type DNA polymerase with high fidelity is often inferior to the PolI type DNA polymerase in terms of extendibility. As to the PCR method using the mixture of the α type DNA polymerase and the PolI type DNA polymerase, the fidelity thereof is inferior to that of DNA polymerase with high fidelity. Thus it is desired to develop DNA polymerase or a DNA amplification system which is excellent in all of the points.
DNA Replication Process
Generally, it is required for initiation of the DNA replication that a double strand structure is unpaired at an origin of the replication. DNA helicase is required for that process. A single strand DNA binding protein is bound to the unpaired DNA to stabilize the single strand. Furthermore, primase works on each chain to synthesize the primer. Subsequently, a replication factor C (RFC) recognizes the primer and binds thereto for inducing a proliferation cell nuclear antigen (PCNA) on the DNA chain. PCNA serves as a clump to fasten the DNA polymerase on the DNA chain. And DNA polymerase complexed with PCNA synthesizes a new chain. In a process of continuous synthesis, the long new chain is synthesized in accordance with the aforementioned manner. In a process of discontinuous synthesis, RNA primer attached to each Okazaki fragment is decomposed with nuclease whereby it is replaced with the DNA chain. Subsequently the fragments are connected with DNA ligase, to complete one new chain (Non-patent Documents 4 and 5).
Pfu-PCNA and RFC
It has been reported that Pyrococcus.furiosus PCNA (hereinafter represented by “Pfu-PCNA” or “PfuPCNA”) has a molecular weight of 28.0 kDa, forms a homotrimer in a similar manner to PCNA in eukaryotic organisms and works by interacting with polymerase (Non-Patent Document 6). Meanwhile, Pyrococcus.furiosus RFC (hereinafter represented by “Pfu-RFC” or “PfuRFC”) has a structure constituted by subunits RFCS and RFCL. The open reading frame for Pfu-RFCS encodes one intein and mature Pfu-RFCS has the molecular weight of 37.4 kDa. Pfu-RFCL has the molecular weight of 55.3 kDa. It has been reported that an addition of Pfu-RFC and Pfu-PCNA remarkably promoted the DNA extension activity of Pfu DNA polymerase in a primer extension analysis (Non-patent Document 7).
Structure and nature of Pfu-PCNA
A crystal structure analysis of Pfu-PCNA was performed (Non-patent Document 8). According to that analysis, a Pfu-PCNA trimer is formed with hydrogen bonds between main chain anti-parallel β strands, βI1 and βD2, of two subunits (T108-K178, T110-E176, R112-E174). Further, an intermolecular ion pair network consisting of acidic and basic amino acid side chains is involved in keeping the trimer structure. There is a report on investigation for two mutants, PfuPCNA (D143A) and PfuPCNA (D143A/D147A), obtained by substituting D143 and D147 with alanine, which is a neutral amino acid, among residues involved in the ion pair network (R82, K84 and R109 in an N terminal region and E139, D143 and D147 in a C terminal region) (Non-patent Document 9). In this report, it has been described that PfuPCNA (D143A) and PfuPCNA (D143A/D147A) are eluted in gel filtration at positions corresponding to monomers, and that the crystal is obtained as not the trimers but V-shaped dimers. The report also describes additional results of measuring the activity in the primer extension analysis. In the report, it is concluded that PfuPCNA (D143A/D147A) did not exhibit a DNA synthesis promoting activity both in the cases of PCNA alone and in combination with RFC, whereas PfuPCNA (D143A) exhibited the DNA synthesis promoting activity regardless of the presence or absence of RFC and, in the case of PCNA alone, showed better result than a wild type PCNA.
KOD-PCNA and RFC
KOD-PCNA (hereinafter represented by “KOD-PCNA” or “KODPCNA”) and KOD-RFC (hereinafter represented by “KOD-RFC” or “KODRFC”) are PCNA and RFC obtained from Themmococcus.kodakaraensis KOD-1 strain.
KOD-PCNA has been reported in Non-patent Document 12. According to this report, KOD-PCNA has 249 residues and its theoretical molecular weight is 28.2 kDa. Pfu-PCNA reported previously also has 249 residues, and 84.3% of amino acid residues in both amino acid sequences are identical. KOD-PCNA as well as Pfu-PCNA keeps all of the conservative regions characteristic for PCNA. Although the crystal structure analysis of KOD-PCNA is not performed, it has been described that it is highly likely that KOD-PCNA forms the homotrimer in the same form as in Pfu-PCNA assumed on the basis of its high homology to Pfu-PCNA.
KOD-RFC has been reported in Non-patent Document 10. According to the report, KOD-RFC takes the same subunit structure consisting of RFCL and RFCS as the other RFCs. KOD-RFCL has the molecular weight of 57.2 kDa. The RFCS gene encodes one intein in the open reading frame, and mature KOD-RFCS has the molecular weight of 37.2 kDa.
The above two documents have also reported the effects when KOD-PCNA and KOD-RFC were added to the DNA synthesis system with KOD-DNA polymerase. In the reports, it has been described that KOD-PCNA alone and KOD-PCNA in combination with KOD-RFC promoted the extension activity in the primer extension experiment, and the “sensitivity” was enhanced when KOD-PCNA was added in the PCR reaction system.
Patent Document 1: U.S. Pat. No. 5,436,149
Patent Document 2: U.S. Pat. No. 5,338,671
Nonpatent Document 1: Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. and Erlich, H. A., Science, 239, 487-491 (1988)
Nonpatent Document 2: Cline, J C Braman, and H H Hogrefe Nucl. Acids Res., 24, 3546-3551 (1996)
Nonpatent Document 3: Barnes, W. M., Proc. Natl. Acad. Sci., 91, 2216-2220 (1994)
Nonpatent Document 4: Waga, S, and Stillman, B. Annu. Rev. Biochem., 67, 721-751 (1998)
Nonpatent Document 5: Kornberg, A. and Baker, T. A., DNA replication, 2nd ed. W.H. Freeman, New York. (1992)
Nonpatent Document 6: Cann et al., J. Bacteriol., 181, 6591-6599 (1999)
Nonpatent Document 7: Cann et al., J. Bacteriol., 183, 2614-2623 (2001)
Nonpatent Document 8: Matsumiya et al., Protein Sci., 10, 17-23 (2001)
Nonpatent Document 9: Matsumiya et al., Protein Sci., 12, 823-831 (2003)
Nonpatent Document 10: Kitabayashi et al. Biosci Biotechnol Biochem., November; 67(11): 2373-2380 (2003)
Nonpatent Document 11: Takagi et al., Appl. Environ. Microbiol., November; 63(11): 4504-4510 (1997)
Nonpatent Document 12: Kitabayashi et al., Biosci. Biotechnol. Biochem., October; 66(10): 2194-2200 (2002)